BIBW22 BS, potent multidrug resistance-reversing agent, binds directly to P-glycoprotein and accumulates in drug-resistant cells.

The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.